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Signal peptidase complex subunit 1 participates in the assembly of hepatitis C virus through an interaction with E2 and NS2.

Suzuki R, Matsuda M, Watashi K, Aizaki H, Matsuura Y, Wakita T, Suzuki T - PLoS Pathog. (2013)

Bottom Line: Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired.SPCS1 was found to interact with both NS2 and E2.Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan. ryosuke@nih.go.jp

ABSTRACT
Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a hydrophobic, transmembrane protein that is required not only for NS2-NS3 cleavage, but also for infectious virus production. To identify cellular factors that interact with NS2 and are important for HCV propagation, we screened a human liver cDNA library by split-ubiquitin membrane yeast two-hybrid assay using full-length NS2 as a bait, and identified signal peptidase complex subunit 1 (SPCS1), which is a component of the microsomal signal peptidase complex. Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired. Propagation of Japanese encephalitis virus was not affected by knockdown of SPCS1, suggesting that SPCS1 does not widely modulate the viral lifecycles of the Flaviviridae family. SPCS1 was found to interact with both NS2 and E2. A complex of NS2, E2, and SPCS1 was formed in cells as demonstrated by co-immunoprecipitation assays. Knockdown of SPCS1 impaired interaction of NS2 with E2. Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles.

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Effect of SPCS1 knockdown on the processing of HCV structural proteins and secretion of host proteins.(A) Core-NS2 polyprotein was expressed in KD#31 cells or parental Huh-7 cells. Core, NS2, SPCS1, and actin were detected by immunoblotting 2 days post-transfection. (B) Expression constructs of NS2 and NS2/3 proteins. His to Ala substitution mutation at aa 956 in NS2 is indicated by an asterisk. Gray circles and bold lines indicate FLAG-tag and the spacer sequences, respectively. Positions of the aa residues are indicated above the boxes. (C) Effect of SPCS1 knockdown on processing at the NS2/3 junction. Huh-7 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 30 nM, and then transfected with plasmids for FLAG-NS2, F-NS2-3, or F-NS2-3 with a protease-inactive mutation (H956A). NS2 in cell lysates was detected by anti-FLAG antibody 2 days post-transfection. Arrowhead indicates unprocessed NS2-3 polyproteins. (D) Effect of SPCS1 knockdown on the secretion of apoE. Huh7.5.1 cells were transfected with SPCS1 siRNAs or control siRNA at a final concentration of 20 nM, and apoE in the supernatant and SPCS1 and actin in the cells were detected 3 days post-transfection. (E) Effect of SPCS1 knockdown on the secretion of albumin. Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA, and albumin in the culture supernatants at 2 and 3 days post-transfection was measured by ELISA.
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ppat-1003589-g004: Effect of SPCS1 knockdown on the processing of HCV structural proteins and secretion of host proteins.(A) Core-NS2 polyprotein was expressed in KD#31 cells or parental Huh-7 cells. Core, NS2, SPCS1, and actin were detected by immunoblotting 2 days post-transfection. (B) Expression constructs of NS2 and NS2/3 proteins. His to Ala substitution mutation at aa 956 in NS2 is indicated by an asterisk. Gray circles and bold lines indicate FLAG-tag and the spacer sequences, respectively. Positions of the aa residues are indicated above the boxes. (C) Effect of SPCS1 knockdown on processing at the NS2/3 junction. Huh-7 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 30 nM, and then transfected with plasmids for FLAG-NS2, F-NS2-3, or F-NS2-3 with a protease-inactive mutation (H956A). NS2 in cell lysates was detected by anti-FLAG antibody 2 days post-transfection. Arrowhead indicates unprocessed NS2-3 polyproteins. (D) Effect of SPCS1 knockdown on the secretion of apoE. Huh7.5.1 cells were transfected with SPCS1 siRNAs or control siRNA at a final concentration of 20 nM, and apoE in the supernatant and SPCS1 and actin in the cells were detected 3 days post-transfection. (E) Effect of SPCS1 knockdown on the secretion of albumin. Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA, and albumin in the culture supernatants at 2 and 3 days post-transfection was measured by ELISA.

Mentions: Since SPCS1 is a component of the signal peptidase complex, which plays a role in proteolytic processing of membrane proteins at the ER, it may be that SPCS1 is involved in processing HCV proteins via interacting with ER membranes. To address this, the effect of SPCS1 knockdown on the processing of HCV precursor polyproteins in cells transiently expressing the viral Core-NS2 region was analyzed. Western blotting indicated that properly processed core and NS2 were observed in KD#31 cells as well as Huh-7 cells (Fig. 4A). No band corresponding to the unprocessed precursor polyprotein was detected in either cell line (data not shown). We also examined the effect of SPCS1 knockdown on the cleavage of the NS2/3 junction mediated by NS2/3 protease. Processed NS2 was detected in both cell lines with and without SPCS1 knockdown, which were transfected with wild-type or protease-deficient NS2-3 expression plasmids (Fig. 4B & C).


Signal peptidase complex subunit 1 participates in the assembly of hepatitis C virus through an interaction with E2 and NS2.

Suzuki R, Matsuda M, Watashi K, Aizaki H, Matsuura Y, Wakita T, Suzuki T - PLoS Pathog. (2013)

Effect of SPCS1 knockdown on the processing of HCV structural proteins and secretion of host proteins.(A) Core-NS2 polyprotein was expressed in KD#31 cells or parental Huh-7 cells. Core, NS2, SPCS1, and actin were detected by immunoblotting 2 days post-transfection. (B) Expression constructs of NS2 and NS2/3 proteins. His to Ala substitution mutation at aa 956 in NS2 is indicated by an asterisk. Gray circles and bold lines indicate FLAG-tag and the spacer sequences, respectively. Positions of the aa residues are indicated above the boxes. (C) Effect of SPCS1 knockdown on processing at the NS2/3 junction. Huh-7 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 30 nM, and then transfected with plasmids for FLAG-NS2, F-NS2-3, or F-NS2-3 with a protease-inactive mutation (H956A). NS2 in cell lysates was detected by anti-FLAG antibody 2 days post-transfection. Arrowhead indicates unprocessed NS2-3 polyproteins. (D) Effect of SPCS1 knockdown on the secretion of apoE. Huh7.5.1 cells were transfected with SPCS1 siRNAs or control siRNA at a final concentration of 20 nM, and apoE in the supernatant and SPCS1 and actin in the cells were detected 3 days post-transfection. (E) Effect of SPCS1 knockdown on the secretion of albumin. Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA, and albumin in the culture supernatants at 2 and 3 days post-transfection was measured by ELISA.
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ppat-1003589-g004: Effect of SPCS1 knockdown on the processing of HCV structural proteins and secretion of host proteins.(A) Core-NS2 polyprotein was expressed in KD#31 cells or parental Huh-7 cells. Core, NS2, SPCS1, and actin were detected by immunoblotting 2 days post-transfection. (B) Expression constructs of NS2 and NS2/3 proteins. His to Ala substitution mutation at aa 956 in NS2 is indicated by an asterisk. Gray circles and bold lines indicate FLAG-tag and the spacer sequences, respectively. Positions of the aa residues are indicated above the boxes. (C) Effect of SPCS1 knockdown on processing at the NS2/3 junction. Huh-7 cells were transfected with SPCS1 siRNA or control siRNA at a final concentration of 30 nM, and then transfected with plasmids for FLAG-NS2, F-NS2-3, or F-NS2-3 with a protease-inactive mutation (H956A). NS2 in cell lysates was detected by anti-FLAG antibody 2 days post-transfection. Arrowhead indicates unprocessed NS2-3 polyproteins. (D) Effect of SPCS1 knockdown on the secretion of apoE. Huh7.5.1 cells were transfected with SPCS1 siRNAs or control siRNA at a final concentration of 20 nM, and apoE in the supernatant and SPCS1 and actin in the cells were detected 3 days post-transfection. (E) Effect of SPCS1 knockdown on the secretion of albumin. Huh7.5.1 cells were transfected with SPCS1 siRNA or control siRNA, and albumin in the culture supernatants at 2 and 3 days post-transfection was measured by ELISA.
Mentions: Since SPCS1 is a component of the signal peptidase complex, which plays a role in proteolytic processing of membrane proteins at the ER, it may be that SPCS1 is involved in processing HCV proteins via interacting with ER membranes. To address this, the effect of SPCS1 knockdown on the processing of HCV precursor polyproteins in cells transiently expressing the viral Core-NS2 region was analyzed. Western blotting indicated that properly processed core and NS2 were observed in KD#31 cells as well as Huh-7 cells (Fig. 4A). No band corresponding to the unprocessed precursor polyprotein was detected in either cell line (data not shown). We also examined the effect of SPCS1 knockdown on the cleavage of the NS2/3 junction mediated by NS2/3 protease. Processed NS2 was detected in both cell lines with and without SPCS1 knockdown, which were transfected with wild-type or protease-deficient NS2-3 expression plasmids (Fig. 4B & C).

Bottom Line: Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired.SPCS1 was found to interact with both NS2 and E2.Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan. ryosuke@nih.go.jp

ABSTRACT
Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a hydrophobic, transmembrane protein that is required not only for NS2-NS3 cleavage, but also for infectious virus production. To identify cellular factors that interact with NS2 and are important for HCV propagation, we screened a human liver cDNA library by split-ubiquitin membrane yeast two-hybrid assay using full-length NS2 as a bait, and identified signal peptidase complex subunit 1 (SPCS1), which is a component of the microsomal signal peptidase complex. Silencing of endogenous SPCS1 resulted in markedly reduced production of infectious HCV, whereas neither processing of structural proteins, cell entry, RNA replication, nor release of virus from the cells was impaired. Propagation of Japanese encephalitis virus was not affected by knockdown of SPCS1, suggesting that SPCS1 does not widely modulate the viral lifecycles of the Flaviviridae family. SPCS1 was found to interact with both NS2 and E2. A complex of NS2, E2, and SPCS1 was formed in cells as demonstrated by co-immunoprecipitation assays. Knockdown of SPCS1 impaired interaction of NS2 with E2. Our findings suggest that SPCS1 plays a key role in the formation of the membrane-associated NS2-E2 complex via its interaction with NS2 and E2, which leads to a coordinating interaction between the structural and non-structural proteins and facilitates the early step of assembly of infectious particles.

Show MeSH
Related in: MedlinePlus